During development, the olfactory sensory neuron (OSN) axons navigate through the mesenchyme, coalesce with other axons expressing the same odorant receptor, and converge into a glomerulus in a topographically defined region of the olfactory bulb (OB). Although considerable effort has been made, the mechanisms regulating these events are not well understood. My long- term goal is to understand the mechanisms mediating this progression. Currently I am interested in the role that functional activity has in these events. This application focuses on the contribution of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity to olfactory development. The HCN channel current Ih was first characterized in cardiac pacemaking activity; the channels are found extensively in the central nervous system. HCN channels are gated by cAMP, which can affect channel kinetics depending on which of the four HCN subunits (HCN1-4) are present. The cyclic nucleotide binding domain of HCN channels make them good candidates for affecting the cAMP activity recently implicated in olfactory system development (Imai et al., 2006, Zou et al., 2007, Chesler et al., 2007). The following aims are designed to determine the role of HCN channels in OSN axon outgrowth, growth cone activity, coalescence and glomerular formation. We previously showed an upregulation of the HCN2 subunit during development (Mobley et al., 2010). Specific Aim 1 examines the mechanisms underlying the role of HCN2, which is strongly modulated by cAMP, in development. I will employ in utero electroporation of the olfactory epithelium to introduce a plasmid expressing GFP and HCN2 shRNA targeting OSNs. This original technique adapted to the nasal cavity will enable visualization of OSN axon behavior in vivo and may have a lasting influence on future studies of OSN dynamics. Specific Aim 2 examines the role of HCN channels and membrane potential in growth cone lamellipodia formation. Guidance signal-induced cAMP signaling is important for OSN axon growth cone targeting and coalescence. Elsewhere in brain cyclic nucleotide signaling is implicated in growth cone dynamics, though the role of HCN channels has not been explored (Ming et al., 1997; Maritan et al., 2009; Murray et al., 2009). Thus, our analyses of HCN mediated growth cone dynamics in OSNs would not only impact olfactory system development but on developmental mechanisms throughout the brain. Specific Aim 2 will challenge OSNs in vitro with membrane potential changes and HCN channel blockers to measure changes in growth cone lamellipodia and filopodia. Demonstrating a role for HCN channels in growth cone dynamics would impact a much larger field than olfactory research and provide new avenues for investigating activity dependent mechanisms of olfactory system development. The results of these studies may provide mechanisms for OSN axon targeting during development such as age-dependent subunit regulation and membrane potential-induced growth cone dynamics. The experiments proposed here may have broad implications for Ih channelopathies including sinus bradycardia, epilepsy, and peripheral neuropathic pain. PUBLIC HEALTH RELEVANCE: This proposal seeks to determine how hyperpolarization-activated cyclic nucleotide-gated (HCN) channels influence development of the olfactory system. This study will expand our understanding of how activity- dependent mechanisms influence axon growth cone dynamics, coalescence and glomerular formation during development, and may apply to the study of olfactory deficiencies such as Kallman syndrome. In turn, these results may apply broadly to cardiac pacemaking, sinus bradycardia, some forms of epilepsy and neuropathic pain, in which HCN channels are implicated.